CN1646679A - Substrate and method for measuring the electrophysiological properties of cell membranes - Google Patents
Substrate and method for measuring the electrophysiological properties of cell membranes Download PDFInfo
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- CN1646679A CN1646679A CNA038085569A CN03808556A CN1646679A CN 1646679 A CN1646679 A CN 1646679A CN A038085569 A CNA038085569 A CN A038085569A CN 03808556 A CN03808556 A CN 03808556A CN 1646679 A CN1646679 A CN 1646679A
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Abstract
The present invention relates to a substantially planar substrate for use in patch clamp analysis of the electrophysiological properties of a cell membrane comprising a glycocalyx, wherein the substrate comprises an aperture having a rim, the rim being adapted to form a gigaseal upon contact with the cell membrane. The invention further provides a method of making such a substrate and method for analysing the electrophysiological properties of a cell membrane comprising a glycocalyx.
Description
Technical field
The present invention is by setting up a kind of electrophysiologicalmeasurements measurements configuration, cytolemma wherein forms the high resistance sealing coat that surrounds potential electrode, it can be determined and monitoring stream is crossed the electric current of cytolemma, thereby a kind of substrate and method of determining and/or monitoring the ionic channel electricity physiological property that contains the ionic channel structure is provided, and this ionic channel structure contains the lipid membrane structure just like cell usually.More specifically say, the present invention relates to a kind of substrate and a kind of method, be used to analyze the cytolemma character that comprises glycocalix.This substrate normally is used for studying the part of the equipment of cytolemma electric process, such as the patch clamp technique equipment that is used to study microbial film intermediate ion passage.
Background technology
Foreword
Make the diaphragm electrical isolation and the universal of the ionic channel of research in this diaphragm under the voltage clamp condition, see the article " TheExtracellular Patch Clamp; A Method For Resolving CurrentsThrough Individual Open Channels In Biological Membranes " of Neher, Sakmann and Steinback (1978), Pflueger Arch.375; 219-278.Have been found that and contain vagusstoff facing to muscle cell membrane extruding (acetylcholine, glass pipet ACh) can be seen that electric current is discontinuous to beat, and these are beated and are attributable to the switch of Ach activated ionic channel.But these researchists are subjected to the restriction of the following fact in their work: the resistance of sealing coat between glass pipet and the film (10-50M Ω), and more much smaller than the resistance (10G Ω) of passage.Electrical noise from this sealing coat produces is inversely proportional to resistance, so electrical noise is big must be enough to the electric current that shield flow is crossed ionic channel, and the electric conductivity of ionic channel is more much smaller than Ach passage.Owing to produce the big electric current that flows through sealing coat, also stop voltage clamp in the transfer pipet on the value different with this liquid.
Find afterwards, by using fire to glass pipet polishing and air-breathing to transfer pipet inside, can obtain the very sealing coat of high resistance (1 to 100G Ω) at cell surface, thereby noise is reduced an order of magnitude, so that reach the degree that biological corridor that great majority will study can be studied, greatly expanded the voltage range that these researchs can be carried out simultaneously.This improved sealing coat is called as " gigohm nurse sealing coat (" gigaseal ") ", and this kind transfer pipet is called as " diaphragm transfer pipet (" patch pipette ") ".The explanation of " gigohm nurse sealing coat " in more detail, as seen O.P.Hamill, A.Marty, E.Neher, B.Sakmann, and the article of F.J.Sigworth (1981): " Improved patch-clamp techniques for highresolution current recordings from cells and cell-free membranepatches ", Pflueger Arch.391; 85-100.Because they develop the work of patch clamp technique, Neher and Sakmann obtain biomedical Nobel prize in 1991.
Ionic channel is the protein of transmembrane, and it impels mineral ion to cross over transporting of cytolemma.Ionic channel participates in all processes, as the electromotive force of generation and time action, the transmission of cynapse, the secretion of hormone, contraction of muscle or the like.Many medicaments produce their specific effects by the adjusting of ionic channel.Example comprises: the anti-epileptic mixture, and as Phenytoin Sodium Salt (phenytoin) and lamotrigine, these medicaments are blocked Na+ passage in the cerebral tissue relevant with voltage; Antihypertensive drug, as nifedipine (nifedipine) and diltiazem (diltiazem), these medicines are blocked Ca2+ passage in the smooth muscle cell relevant with voltage; Discharge stimulant with Regular Insulin, as glibenclamide and first sulphur fourth arteries and veins (tolbutamide), the K+ passage in the pancreas of these stimulant blocking-up adjustment ATP.Except the adjusting that chemically causes ion channel activity, patch clamp technique can make scientist handle the passage relevant with voltage.These technology comprise the polarity of adjusting electrode in the diaphragm transfer pipet and change the brinish component, the level of free ion in the restraining liquor.
Patch clamp technique
Patch clamp technique is represented the main development of biological and medical science, because its energy measurement flows through the ion of single ionophorous protein matter, can also study the activity of single ion channel response drug dose.Briefly, in the patch clamp operation of standard, use thin glass pipet (about 0.5～2 μ m diameter).The point of this diaphragm transfer pipet is facing to the surface of cell membrane extruding.The transfer pipet point is closing cell's film tightly, and isolates a small amount of ionophorous protein matter population that is subjected to the restriction of the transfer pipet mouth of pipe.The activity of these passages can perhaps, can be made rupture of diaphragm by indivedual measure (" single channel recordings "), the channel-active of energy measurement whole cell membrane (" full cell configuration ").By in transfer pipet, applying negative pressure, make membranolysis, can be for implementing the high conductivity that full cell measurement acquisition enters cell interior.
Gigohm nurse sealing coat
As mentioned above, the patch clamp of single channel current is measured, an important requirement is to set up high-resistance sealing coat between cytolemma and the little transfer pipet point of glass, so that move in the space of restriction ion between two surfaces.Usually, require to surpass the resistance of 1G Ω, therefore, this physics zone of action is called " gigohm nurse sealing coat ".
The formation of gigohm nurse sealing coat requires cytolemma and transfer pipet glass to come in close proximity to each other.Therefore, the distance of flanking cell in tissue, or the distance of cultured cells and their substrate generally when the magnitude of 20-40nm (Neher, 2001), can be predicted the distance between cytolemma and the transfer pipet glass, should be at Angstr m (promptly 10
-10M) scope.Still do not know the physicochemical property of gigohm nurse sealing coat.But gigohm nurse sealing coat can form between cytolemma and various glass, comprises quartz, silicated aluminum and silication boron (Rae and Levis, 1992), and the particular chemical component that shows glass is not crucial.
Membrane structure
Cytolemma is made of phospholipid bilayer, and sandwich glycoprotein, glycoprotein have multiple function, comprises the acceptor as all ingredients.These intermembranous glycoprotein generally include peptide and sugar respectively accounts for half, from film, stretch out, enter extracellular space, form what is called " glycocalix " layer that surrounds phospholipid bilayer, the height of glycocalix layer is 20 to 50nm, and foundation and phospholipid bilayer adjacency be full of electrolytical compartment (see figure 1).Therefore, glycocalix forms hydrophilic and electronegative zone, constitutes the gap between cell and its aqueous environments.
Cytoskeleton and glycocalix
Below cytolemma is the molecule of cytoskeleton, muscle fibrin silk screen, spectrin, articulin and multiple other macrostructures.Cytoskeletal a kind of vital role is that some integral membrane albumen and glycoprotein are anchored on the film internal fixing position.But, believe be clipped in intermembranous glycoprotein in some limit (at lipid microcell or " raft "; Simons and Toomre, 2000 are seen in comment) in, can freely in phospholipid bilayer, be displaced sideways.Really, this arrangement is described to " as the protein iceberg in the lipid ocean ".
The effect that glycocalix forms gigohm nurse sealing coat
In the patch clamp method of routine, the initial contact point of glass pipet point (the about 100nm of its wall thickness) and cell relates to glycocalix.The estimation of resistance is represented by the 150mM ionogen that comprises in the predetermined gap between glass surface and the lipid film, by the height of glycocalix (as 20 to 40nm), obtains 20-60M Ω.This estimation and experimental observation unanimity at the smooth surface quartz of coating TEOS (Triethyloxysilane, triethyl silicyl oxide) cake core, the resistance that this kind TEOS cake core produces as a rule is in 40M Ω magnitude (or only reach G Ω 4%).In this estimation, suppose between lipid film and approximate cylindricality glass surface to have ionogen the about 1 μ m of this cylindricality glass surface diameter, the about 3-10 μ of length m.Aspirate gently subsequently transfer pipet (＜20hPa), further increase resistance, obtain gigohm nurse sealing coat under the ideal situation.The formation of gigohm nurse sealing coat can take place rapidly on time scale 0.1 to 10s, also may be the process of spinning out, and only finishes after several times increase swabbing pressure in succession.The time course that gigohm nurse sealing coat forms has reflected that glycoprotein by being displaced sideways in " liquid crystal " phospholipid bilayer, is excluded from physics zone of action (film/transfer pipet).In other words, owing to put on the negative hydrostatic pressure of transfer pipet, facing to glass surface (hydrophilic silanol family) extruding phospholipid bilayer (the hydrophilic cartridge of phosphatide), glycocalix, promptly the glycoprotein unit is extruded the zone of action.
But this resistance increase process only advances to and forms accurate gigohm nurse sealing coat (0.5 to 1G Ω) sometimes.By experience, apply big (50-70mV to transfer pipet this moment; Penner, 1995) negative potential, last resistance is increased, terminate on the gigohm nurse sealing coat.With regard to glycocalix, a kind of observations in back can be interpreted as, and the negative transfer pipet electromotive force that the negative live zone of glycoprotein is applied in drives and moves along side direction.Act on electric field (E) intensity on the glycoprotein, promptly from transfer pipet chamber lumen to the electric field that surrounds liquid, can be considered to:
Wall thickness (x) 100nm that supposes the transfer pipet point with the electromotive force (V) that puts on transfer pipet is-70mV.
The conventional transfer pipet and the contrast of planar substrates
Recently planar substrates (as silicon) has been introduced in the development in the patch clamp method, replaces the conventional little transfer pipet of glass (as seeing WO 01/25769 and Mayer, 2000).
Between plane silicon base chip and viable cell, form the trial of gigohm nurse sealing coat, be proved to be problematic (for example seeing Mayer, 2000).But, do not comprise at synthetical and obtain gigohm nurse sealing coat between the phosphatide vesicle of outside glycocalix, achieve success.This discovery shows that in the forming process of gigohm nurse sealing coat, glycocalix plays crucial vital role.
Therefore, exist and improve planar substrates, make it to be suitable for the demand of the electrophysiological patch clamp research of cytolemma, this planar substrates allows to form gigohm nurse sealing coat with the cytolemma that contains glycocalix.
Summary of the invention
The invention provides a kind of substrate and method of optimization, under the full-scale condition that cell and cytolemma are influenced, be used for determining and/or monitoring stream is crossed and contained structure, particularly contain the electric current of ionic channel of the membrane structure of glycocalix.Therefore, the data of using substrate of the present invention and method to obtain, such as, the result that influences as various test compound thing cell membrane, the variation that ion channel activity is taken place, the influence of can be as inherent but not introducing by measuring system artificially and credible, and can be as studying under the relevant specified criteria effective basis of the electroconductibility of cytolemma or capacitive electrophysiological phenomena.
Should be pointed out that when this specification sheets uses the term of " cell " or " cytolemma " literary composition before and after it depends on usually may use any another to contain the ionic channel of structure, contains the ionic channel of lipid film such as another, or contains the ionic channel of artificial membrane.
As mentioned above, the patch clamp of single channel current is measured, an important requirement is to set up high-resistance gigohm nurse sealing coat between cytolemma and substrate.Forming the key factor of gigohm nurse sealing coat, is the degree of approach of cytolemma to substrate, and it depends on the size of zone of action between cytolemma and the substrate again.
Physics contact area between cytolemma and the planar silicon chip with smooth circular funnel-form aperture (about 1 μ m wide contact edge; See the figure of Fig. 2 right-hand side), than form between cytolemma and the little transfer pipet of glass (about 100nm is wide; See the figure of Fig. 2 left-hand side) much bigger.This result causes the force rate transfer pipet configuration of per unit area in the chip significantly to reduce, and the glycoprotein quantity that is clipped in the middle on contact area becomes more, effectively prevents phospholipid bilayer and forms the Angstr m distance that requires between the requisite substrate surface of gigohm nurse sealing coat.
The present invention is by providing a kind of planar substrates (as silica-based chip), be fit to patch clamp research for cytolemma electricity physiological property, find the approach that addresses this problem, this planar substrates is exclusively used in provides the contact area of reduction with cytolemma, thereby has improved the formation of gigohm nurse sealing coat.
Therefore, a first aspect of the present invention, provide a kind of flat basically substrate, be used to comprise the electric physiological property patch clamp analysis of the cytolemma of glycocalix, substrate wherein comprises the aperture of stipulating this aperture with the edge, this edge is fit to form gigohm nurse sealing coat when contacting with cytolemma.
In a preferred embodiment, this substrate is silica-based chip.
In the present context, term gigohm nurse sealing coat typically refers to the sealing coat of lG ohm at least, and should size generally be the minimum target of sealing coat, but to the Super-Current Measurement of some type, enough with lower value as threshold value.
" glycocalix ", we are meant the layer that is respectively accounted for half foundation by peptide and sugar, the glycoprotein from the phospholipid bilayer of cytolemma stretches out, and enters extracellular space.
More preferably, this edge surpasses the height of glycocalix on the phospholipid bilayer of cytolemma from the height of substrate plane projection.Preferablely be that this edge stretches out at least 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm or 100nm at least at least at least at least at least at least at least at least on substrate plane.
Favourable way is, the shape at this edge can make the physics contact area between substrate and cytolemma become minimum, thus help glycocalix pass and form gigohm nurse sealing coat.
The one skilled in the art is obviously clear, and this edge can be any suitable cross-sectional shape.For example, the wall at edge can be tapered or be substantially parallel.Equally, some kinds of shapes can be got in the top at edge, and for example, it can be dome-shaped, smooth or sharp.In addition, the salient at edge can be substantially perpendicular to, favours or be parallel to the plane of substrate.The parallel edge that stretches out can be located on or near the mouth of aperture, perhaps more in depth is positioned at aperture.In general, the width at edge is between 10 to 200nm.
In addition, this edge can be by the interruption-forming of aperture itself, rather than is formed by salient.The mouth of aperture can have 5 to 100nm radius-of-curvature, has 45 to 90 degree acute angles.
The one skilled in the art is obviously also clear, and the size of this aperture should not allow complete cell by this planar substrates.
The length of aperture (being the degree of depth) is preferably between 2 to the 30 μ m, for example between 2 to 20 μ m, 2 to 10 μ m or 5 to 10 μ m.
To the best hole diameter that best gigohm nurse sealing coat forms and full cell is set up, relevant with the particular cell types of using.Hole diameter is comparatively favourable in the scope of 0.5 to 2 μ m.
The parts that substrate of the present invention normally uses in the equipment are so that realize the measurement that lipid film such as cytolemma intermediate ion transmit the certain electric physiological property.
The design of this equipment, purpose provide the device of finishing a large amount of Individual testwas at short notice.This realizes that by a kind of micro-system is provided this micro-system has a plurality of test compartments (the marginate aperture that promptly is used for exposing cell), and the position that comprises integrated potential electrode is respectively arranged, and suitable specimen source also is provided.Each test compartment can comprise: the device of make cellular localization, set up gigohm nurse sealing coat, the position of gigohm nurse sealing coat having been set up in selection; The electrode of measuring; With one or more reference electrodes.Thereby can test in the compartment at each and implement independently experiment and, control the preparation and the measurement of all experiments from central control unit computer for example.Because the little size of test compartment, the present invention can use only very a spot of carrier liq and specimen, can finish measurement.
Substrate of the present invention can be made with the material of any suitable processing of wafers technology, for example silicon, plastics, pure quartz and other glass, as quartz and PyrexTM, or mixed that one or more are selected from the quartz of following hotchpotch: Be, Mg, Ca, B, Al, Ga, Ge, N, P, As.Silicon is preferred substrate material.
In a preferred embodiment of first aspect present invention, the wall of substrate surface and/or aperture has applied the very suitable material of setting up sealing coat with cytolemma.These materials comprise: silicon, plastics, pure quartz and other glass, as quartz and PyrexTM, or mixed that one or more are selected from the oxide compound of the quartz of following hotchpotch: Be, Mg, Ca, B, Al, Ga, Ge, N, P, As and these materials.Substrate is preferably to small part coating silicon oxide.
In another preferred embodiment of first aspect present invention, this planar substrates has first surface part and reciprocal second surface part, this first surface partly has at least a position to be fit to the ionic channel that clamping contains structure, each position comprises marginate aperture and associated potential electrode is arranged, this substrate is loaded with one or more reference electrodes, potential electrode and reference electrode are arranged on each position of aperture and are full of electrolytical compartment, these potential electrode and corresponding reference electrode or reference electrode, when in ionogen, contacting with each other and when being coupled with potential difference between them, these electrodes can be sent ion and another electrode reception ion by an electrode, between them, produce electric current, each position is adapted between the surface portion at the ionic channel that contains structure of this position clamping and this position, high-resistance sealing coat is provided, the sealing coat that this provides, the zone that defines and in ionogen, contact on ionic channel one side that contains structure with potential electrode, separate with the zone of corresponding reference electrode contact with definition on the ionic channel opposite side that contains structure and in ionogen, so, flow through the ion channel current that contains the ionic channel of structure between each electrode that is positioned at each side of substrate, can be determined and/or monitored.
The example of the general design of first aspect present invention preferred embodiment, substrate wherein comprise the entire electrode feature of the marginate aperture of the present invention (but do not comprise), explanation in WO 01/25769.
A second aspect of the present invention provides a kind of method of making substrate according to first aspect present invention, and present method comprises the steps:
(i) provide the substrate template;
(ii) in template, form aperture; With
(iii) form the edge around aperture.
The most handy silicon micro production of this substrate technology " Madou, M., 2001 " is made.
The one skilled in the art should be understood that, step (ii) and (iii) can be implemented (promptly separation steps) in time in order, also can implement simultaneously.
Favourable way is that step (ii) comprises uses inductive couple plasma (ICP) deep ion etching treatment procedure (DE 4241045 for Laermer F.And Schilp, A.) to form aperture.
When needs formation is substantially perpendicular to the salient of substrate plane, present method comprises an intermediate steps, this step has direction ground and a side of etch substrate front selectively, mask layer on one side of substrate front is removed, and further the distance of aforementioned salient is continued to enter following substrate.
Because silicon has etch-rate faster than mask material, the result is that mask material will be stayed the aperture inboard, and protrude from substrate.Can apply the coating on whole surface subsequently.
When needs form basically the salient in substrate plane, present method comprises an intermediate steps, this step is used Inductive Coupled Plasma (ICP) etching that forms micropore, or Advanced Silicon Etch (ASE), these methods are characterised in that etching of repetition alternative and passivation step, can produce the pit that some point to microstome.By suitable adjustment process parameters, can in salient plane, edge, obtain pit inwards.
Still the same, can adopt the coating on whole surface subsequently.
Usually, before or after aperture and/or edge formation, present method also comprises coated substrates surface (for example using silicon oxide).In addition, step is (iii) implemented in the substrate coating.
Coating can be with plasma-reinforced chemical vapour deposition (PECVD) and/or with low pressure chemical vapor deposition (LPCVD).
The first aspect present invention preferred embodiment, substrate wherein comprises Integrated electrode, can be by the explanation manufacturing among the WO 01/25769).
A third aspect of the present invention provides a kind of method, is used to analyze the electric physiological property of the cytolemma that contains glycocalix, and present method comprises:
(i) provide the substrate that the edge aperture is arranged according to first aspect present invention;
(ii) make cytolemma and substrate openings edge contact, so that between cytolemma and substrate, form gigohm nurse sealing coat; With
(iii) measure the electric physiological property of this cytolemma.
In a preferred embodiment of third aspect present invention, provide a kind of method of setting up full cell measurement configuration, be used for determining and/or monitoring the electric physiological property of one or more ionic channels of one or more ionic channels that contain structure, described method comprises the steps:
(i) provide substrate by above-mentioned definition;
(ii) provide carrier liq on one or more apertures, described carrier liq comprises one or more ionic channels that contains structure;
(iii) at least one ionic channel that contains structure is positioned on the aperture of respective amount;
(iv) by between the potential electrode and reference electrode relevant with a certain position (being aperture), be continuously applied fixed first potential difference, check the ionic channel that contains structure of this position clamping and high resistance sealing coat between this position surface portion (being the edge) of high resistance sealing coat is provided; Monitoring stream is crossed first electric current between described potential electrode and the described reference electrode; With described first electric current and predetermined threshold value current ratio, and,, can approve so if this first electric current only is the predetermined threshold value electric current at the most, between the ionic channel that contains structure and this position surface portion, this position has acceptable sealing coat; With
(v) set up full cell configuration at the position of approval.
Thus, flow through ionic channel the 3rd electric current of the ionic channel that contains structure between potential electrode and the reference electrode, can determine and/or monitor.
By or initiatively or passive device, can guide the ionic channel that contains structure (as cell) in the solution into a certain position.When this contained the ionic channel of structure and aperture edge contact, the surface in contact at this position formed high resistance sealing coat (gigohm nurse sealing coat), so can be with the electric physiological property of electrode measurement ionic channel.This electric physiological property can be by being surrounded, contain structure by gigohm nurse sealing coat the electric current of membrane portions of ionic channel.
The acquisition of full cell configuration can apply a series of second potential difference pulses between potential electrode and reference electrode relevant with each approval position; Monitoring stream is crossed second electric current between potential electrode and the reference electrode; With when described second electric current surpasses predetermined threshold, interrupt this second potential difference pulses row, thereby make the ionic channel partial rupture that contains structure near potential electrode.
In addition, the acquisition of full cell configuration can interact with the aperture that forms substrate by making the ionic channel that contains structure near the part of potential electrode.
Should be pointed out that in style of writing of the present invention " full cell configuration " speech not only refers to following configuration, in this configuration, in the measuring point, whole cell contacts with substrate, and be punctured, or, can electrically contact, but also refer to following configuration with cell interior by the aperture that forms substrate, in this configuration, the fragment that cuts cytolemma is arranged, and the outside surface of film " is faced up ", towards the specimen that will apply.
Because the potential electrode relevant with a certain position can be one of a plurality of electrodes on the substrate, and the ionic channel that contains structure can be one of many passages in the solution, therefore can obtain the measurement structure of many preparations like this on substrate.A kind of typical measurement comprises to this structure and adds special specimen, and for this reason, each is measured to construct with other measurement structure and separates, and avoids the mixing of specimen and the electricity between each structure to lead.
In the use, the cell carrier liquid and the cell that add to substrate one of can be as follows implemented.In a preferred embodiment, the test compartment can enter from above, and can be by adjusting or liquor-transferring system vector supplier liquid and cell on each test compartment.Can use such as ink jetting head or the first-class system of bubble jet printer.Another kind of may, be the air-breathing adjustment device of nQUAD or any other be fit to the to batch adjustment/liquid-transfering device of low dose of liquid.In addition, carrier liq and cell, be do as a whole be applied to on-chip (for example, by the carrier liq that contains cell is poured on the substrate, or substrate immersed in this carrier liq that contains cell), provide carrier liq and cell to each test compartment in view of the above.Because carrier liq and subsequent specimen volume are little as only to reach and receive liter, evaporation of water may become problem.Therefore, relevant with specific volume, the processing of liquid on the substrate is preferably in the atmosphere of high high humidity and carries out.
In another embodiment, cell is directly cultivated on substrate, is immersed in the growth medium simultaneously.In this best situation, cell will form uniform individual layer (relevant with the cell type that will grow) on whole surface, but deliberately cause except the zone of unsuitable cell growth.The success of cell cultures on the substrate depends on substrate material strongly.
In yet another embodiment, replace a kind of artificial membrane of being to use of cell with merging ionic channel.This artificial membrane can be located on aperture by making the lipid fritter, makes from the lipid saturated solution.This technology is by Christopher Miller (1986) Ion ChannelReconstitution, and Plenum 1986, p.577 explanation fully.If orifice size is suitable, and polar liquid such as water are all arranged, can on aperture, form lipid bilayer in the aperture both sides.Next step is that the protein ionic channel is merged into this bilayer.This is by the side at bilayer, carries to have that the phosphatide vesicle that merges ionic channel reaches.By for example osmotic gradient, can order about these vesicles and bilayer and merge, in view of the above ionic channel is merged into bilayer.
Obtain excellent contact between cell and the glass pipet, thereby and set up gigohm nurse sealing coat between cell and the transfer pipet point, prove absolutely in the prior art.For cell being dragged to the tip of transfer pipet, and produce essential contact, normally aspirate transfer pipet for obtaining this gigohm nurse sealing coat.But, to planar substrates of the present invention, contacting of cytolemma and typical ultrapure quartz substrate only, the bonding on pair cell and surface and to set up gigohm nurse sealing coat enough.
Location on the aperture of cell in substrate can be realized by electrophoresis, and electrophoresis is that the electric field of electrode pulls to electrode to charged cell.Electronegative cell will be pulled to positive electrode, otherwise also true.The static pulling force also can be used as the guide means of one group of electrode.In addition, in the test compartment, hydrophilic material covers and removes the extra-regional substrate surface of hard-pressed bale confining electrode.Therefore, cell only oneself is bonded in electrode area to them.Can use two kinds of methods of these methods simultaneously, or randomly with around the suitable geometrical shape of the substrate surface of electrode combine, the cell that sinks is guided into electrode.
In addition, the location on the aperture of cell in substrate can be realized by osmosis.
If utilization suction, it pulls to aperture to cell, and sets up being connected between cell and aperture, produces the gigohm nurse sealing coat of and solution separating inboard aperture.The desirable any form of this gigohm nurse sealing coat is as circular, oval or rectangle.Wherein, substrate comprises total electrode, and carrier liq can make cytolemma and reference electrode realize electrically contacting.Cell can be out of shape because of suction, and if controlled, cell extends into the situation (but obstructed small holes) of aperture, may need.
Use substrate of the present invention and method, method that can electricity consumption is measured the activity (single channel recording) of ionic channel in cytolemma, in addition, can make rupture of diaphragm, realizes the measurement (full cell record) of whole cell membrane channel-active.At least in three kinds of modes (all three kinds of modes all are feasible, but different cells may get better with different approach work), can obtain high electricity and lead and enter cell interior, to carry out full cell measurement.
(a) cytolemma can break from aperture one side by suction.Perhaps, perhaps, apply pressure below atmospheric pressure as the rising gradually or the step that strengthen intensity as the short pulse that strengthens intensity.Breaking of film by to giving the response of fixed voltage test pulse, the strong capacity current spike that increases occurs and detects (reflecting total cytolemma electric capacity);
(b) by applying voltage pulse, make film rupture.Perhaps, perhaps, between electrode, apply voltage pulse as the rising gradually or the step that strengthen intensity as the short pulse of (mV is to V) and time length (μ s is to ms) that gain in strength.Form the lipid of the film of common cell, be subjected to the influence of the big strength of electric field of voltage pulse, make the neighborhood division of film in view of the above at electrode.Breaking of film by to giving the response of fixed voltage test pulse, the strong capacity current spike that increases occurs and detects.
(c) infiltrationization of film.Use to form the material (for example antibiotic, as nystatin or amphotericin B) of aperture, for example by in advance these materials being deposited on the position.With by making film rupture different because the combination of infiltration chemoattractant molecule, membrane resistance reduces selectively, obtains effectively controlling through the pair cell voltage of electrode pair.Following this bonded is total electrical resistance that descends gradually and the electric capacity that increases gradually.
The substrate here comprises a plurality of test compartments that respectively comprise aperture, can individually add specimen to each test compartment, and each test compartment can have different specimen.These methods can be used to add carrier liq, without these methods, carrier liq be integrally be added in on-chip.
After measuring cellular localization in the configuration, can measure some kinds of electric physiological properties, as flow through the electric current (voltage clamp) of ionic channel, or contain the electric capacity of the ionic channel of film.Under any situation, should provide suitable electronic measurement circuit.The one skilled in the art should select this suitable metering circuit.
A fourth aspect of the present invention provides the kit of the method for implementing third aspect present invention, and this cover instrument comprises that the substrate of first aspect present invention and one or more are used to carry out the substratum or the reactant of patch clamp research.
This cover instrument preferably includes multiple substrate.
Description of drawings
The present invention is illustrated now with reference to unrestriced example of narration and figure:
Fig. 1 draws attached to the cell on the diaphragm transfer pipet.In gigohm nurse sealing coat district, (shadow region with point of contact between transfer pipet point and the cytolemma is represented) glycoprotein of glycocalix is displaced sideways, and can make between membrane phospholipid bilayer and the transfer pipet directly to contact.
Fig. 2 a and 2b draw, and (Fig. 2 is a) with attached to the cell of substrate (Fig. 2 b) attached to the transfer pipet point.Can think, the zone of action between cytolemma and substrate surface, (Fig. 2 is a) greater than the transfer pipet configuration in substrate configuration (Fig. 2 b).
Fig. 3 is to the resistor group of each expection actual transfer pipet changes in resistance of drawing;
Fig. 4 draws Gigaseal (gigohm nurse sealing coat) success ratio to transfer pipet resistance;
Fig. 5 draws success ratio that full cell sets up (from the gigohm nurse sealing coat of success) to transfer pipet resistance;
Fig. 6 draws with the temporal correlation of the gigohm nurse sealing coat formation of different aperture size, and error bars is represented and average standard deviation;
Fig. 7 draws the cell example that adheres on the planar substrates of cocked bead in the aperture side.The formation district of gigohm nurse sealing coat is subjected to many restrictions;
Fig. 8 a, 8b, 8c and 8d draw four kinds and comprise the different aperture design (mould processing) of cocked bead: vertical edge (8a); Sloping edge (8b); Horizontal edge (8c); And built-in edge (8d).
Fig. 9 draws and does not have cocked bead but the design of enough sharp edges (r=25-100nm) is arranged, so that film/substrate zone of action is reduced to 50-200nm.Little angle of spot hole (θ) is 45 to 90 degree;
Figure 10 a and Figure 10 b are the scanning electron microscope diagrams of substrate, and this substrate is done the surface with ICP and LPCVD and revised, and the slotted hole of band cocked bead is arranged in surface plane; With
Figure 11 is the scanning electron microscope diagram of substrate, and this substrate is done the surface with ICP and LPCVD and revised, and the slotted hole of the cocked bead that stretches out from surface plane is arranged.
Embodiment
For example
The present invention contains in cytolemma in the viable cell diaphragm pincers measurement of glycocalix, to the formation of gigohm nurse sealing coat and the foundation of full cell, has discerned three kinds of important factors:
1. the length of aperture is answered sufficiently long, moves so that stop more resilient cell to pass the aperture when suction.
2. to the formation of gigohm nurse sealing coat and the foundation of full cell, also have best orifice size, this size is relevant with the cell type that will study with the elasticity of cytolemma.
3. the aperture of planar substrates, when near cell surface, Ying Youneng moves the edge definition of glycocalix.
To being discussed below of each factor:
The length of aperture
By the length (being the degree of depth) of the aperture of the thickness of chip definition, also very important.The shortcoming of the design of low long-width ratio (short aperture) is owing to cell inherent elasticity, and when making cellular localization and suction subsequently, cell has the trend of passing the hole.Studies show that this problem can surpass 2 μ m usually by with long aperture, alleviates effectively (data are not shown).
Determining of best orifice size
For determining best orifice size, so that obtain gigohm nurse sealing coat and full cell configuration, we use the diaphragm transfer pipet of various size, have compared the success ratio that obtains them in the standard patch clamp structure.Experiment is carried out adhering to and be immersed on the cover glass HEK293 cell in the sodium Ringer solution.The transfer pipet made from silication boron kapillary (Hilgenberg, Cat No.1403573, L=75mm, OD=1.5 mm, ID=0.87mm, the silk of 0.2mm).Transfer pipet resistance is as the telltale of relative orifice size; Manufacturing has the transfer pipet of expection resistance 0.5,1,2,5,10 and 15M Ω.During measurement, write down actual transfer pipet resistance, and each is organized the average of realistic border transfer pipet resistance, and depart from average standard deviation, these data are shown in Fig. 3.
The success ratio that Fig. 4 draws gigohm nurse sealing coat and full cell is to the dependence of transfer pipet aperture resistance orifice size.Experiment number to each group data is carried out is drawn on the data point.The result shows, the transfer pipet of resistance 5M Ω, and to the foundation of the formation of gigohm nurse sealing coat and full cell, success ratio the best, and resistance is 5 until 15M Ω, success ratio has descended about 20%.Transfer pipet resistance drops to below the 5M Ω more harmful; The success ratio of resistance 2M Ω is lower or 50%, 37% than 5M Ω, and resistance 1M Ω or the following formation that will cause not having basically gigohm nurse sealing coat.
Fig. 5 draws and tests the per-cent of the full cell of formation that provides, and these experiments have all successfully formed gigohm nurse sealing coat (promptly be not counted in and do not reach gigohm nurse sealing coat).Data show that though 5M Ω transfer pipet has the highest full cell success ratio, other orifice size has only lower slightly success ratio.
Transfer pipet resistance is to for reaching the effect of used time of G Ω, also in the row (see figure 6) of investigation.The result shows, the transfer pipet of 2M Ω reaches gigohm nurse sealing coat obviously than 5,10 and the length of 15M Ω.5,10 show with the similar result of 15M Ω transfer pipet, increase orifice size in this scope, do not influence the used time of gigohm nurse sealing coat that reaches.
These results clearly show that the success that gigohm nurse sealing coat forms is relevant with the size of transfer pipet aperture.5M Ω transfer pipet has best orifice size, than its big size (promptly having than low resistance), causes the gigohm nurse sealing coat of success to form remarkable decline.
Though top experiment is to carry out with the conventional little transfer pipet of glass, the result can be extrapolated to the substrate that patch clamp experiments is used.Therefore, these results show, the aperture in the chip system, and in general, energy measurement is not greater than the aperture of 5M Ω transfer pipet.But, less than the various transfer pipets of 5M Ω, still can carry out well, but they are obviously bad.Therefore, make the chip aperture be slightly less than 5M Ω transfer pipet, will be littler than it is strengthened harm.
As if change the transfer pipet orifice size, the formation to full cell does not have much affect.Though being formed in the 5M Ω transfer pipet of full cell is the highest, to the transfer pipet of 2M Ω to 15M Ω, success ratio has only reduction slightly.
Also observe, the transfer pipet aperture size is influential to reaching the used time of G Ω.The transfer pipet of 5M Ω and 15M Ω reaches gigohm nurse sealing coat with the identical time, but the transfer pipet of those 2M Ω, be with long 2.5 to 3 times time.
The microscopic examination of the glass pipet that is used to test discloses, and presents the orifice size of the transfer pipet of 5M Ω resistance, is the magnitude of 0.5-1 μ m.But, expect that best orifice size is relevant with the cell size with cell type.
In the patch clamp experiments of routine, when using as HEK or CHO diaphragm culturing cell, the success ratio that obtains gigohm nurse sealing coat is normally high, usually near 90%.According to top consideration, use the little hole geometry of the conventional transfer pipet point of imitation osculum, be expected to the success ratio that on planar chip, reaches suitable.This geometrical shape comprises the aperture at protrusion 0.5 to 1 μ m edge, side.In addition, the length of aperture (being the degree of depth) preferably surpasses 2 μ m.
The manufacturing of planar diaphragm pincers substrate
The present invention makes the preferred method of planar diaphragm pincers substrate, is that it can make silicon oxide on the Si surface applied, forms high-quality glass surface effectively by usefulness silicon (Si) wafer micro production and treatment process.Preferably, make slotted hole and surface modified with ICP (Inductive Coupled Plasma, inductive couple plasma) and LPCVD (Low Pressure Chemical Vapour Deposition, low pressure chemical vapor deposition).Aperture with length of cocked bead can be made aperture and form cocked bead with RIE (Reaction Ion Etch, reactive ion etching) with IPC, carries out surface modified in conjunction with LPCVD.
(a) example in the plane surface of doing to revise on the surface with ICP and LPCVD, is manufactured with the method (Figure 10 a and Figure 10 b) of the long aperture of cocked bead.
1. Kai Shi substrate: silicon single crystal wafer, crystallization direction＜100 〉.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, is obtained the dark upright opening that degree of depth 1-50 μ m is arranged.
4. at silicon face coating etching mask, this mask should be able to tolerate KOH or TMAH solution.For example, it can be silicon oxide or silicon nitride.
5. the opposite side of wafer (downside) applies photo-resist, and the diaphragm-operated figure that contains perforate in the definition silicon nitride, by the exposure of UV light, is transferred on the photo-resist.
6. at the downside of wafer,, chip etching is fallen by the zone of perforate definition in the photo-resist with suitable graph transfer printing technology.The graph transfer printing technology that this is suitable for example, can be Reaction Ion Etch (RIE, reactive ion etching).
7. in KOH or TMAH solution, the etching of wafer is anisotropic, produces the pyramid perforate at the wafer downside.Etched timing definition wafer upside excess silicon diaphragm-operated thickness.In addition, can mix with boron and define etched termination, can provide gauge control preferably.
8. remove the etching mask on the silicon substrate selectively.
9. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: silicon single crystal wafer.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, is obtained the dark upright opening that degree of depth 1-50 μ m is arranged.
4. the opposite side of wafer (downside) applies photo-resist, and the figure that contains the diaphragm definition, by the exposure of UV light, is transferred on the photo-resist.
5. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), substrate is carried out anisotropic etching, obtain the column perforate at the wafer downside.Etching period has defined wafer upside excess silicon diaphragm-operated thickness.
6. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: the silicon on the spacer of buried oxide layer is arranged, and zone of oxidation is at the following 1-50 μ of upper surface m place, carrier crystal orientation＜100 〉.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, obtain dark upright opening, the degree of depth is until the zone of oxidation of burying underground.
4. at silicon face coating etching mask, this mask should be able to tolerate KOH or TMAH solution.For example, it can be silicon oxide or silicon nitride.
5. the opposite side of wafer (downside) applies photo-resist, and the diaphragm-operated figure that contains perforate in the definition silicon nitride, by the exposure of UV light, is transferred on the photo-resist.
6. at the downside of wafer,, chip etching is fallen by the zone of perforate definition in the photo-resist with suitable graph transfer printing technology.The graph transfer printing technology that this is suitable for example, can be Reaction Ion Etch (RIE, reactive ion etching).
7. in KOH or TMAH solution, the etching of wafer is anisotropic, produces the pyramid perforate at the wafer downside.The zone of oxidation of burying underground will be as the etched termination of this process, and therefore, the thickness of silicon layer upside has defined residue diaphragm-operated thickness.
8. by RIE, moist hydracid (HF) etching or the HF vapor etch of fluoridizing, remove the exposed region of the zone of oxidation of burying underground.Can guarantee contacting between wafer middle and upper part and the bottom perforate like this.
9. remove the etching mask on the silicon substrate selectively.
10. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: the silicon on the spacer of buried oxide layer is arranged, and zone of oxidation is at the following 1-50 μ of upper surface m place.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, obtain dark upright opening, the degree of depth is until the zone of oxidation of burying underground.
4. the opposite side of wafer (downside) applies photo-resist, and the figure that contains the diaphragm definition, by the exposure of UV light, is transferred on the photo-resist.
5. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), wafer is carried out anisotropic etching, obtain vertical cavity at the wafer downside.The zone of oxidation of burying underground will be as the etched termination of this process, and therefore, the thickness of silicon layer upside has defined residue diaphragm-operated thickness.
6. by RIE, moist hydracid (HF) etching or the HF vapor etch of fluoridizing, remove the exposed region of the zone of oxidation of burying underground.Can guarantee contacting between wafer middle and upper part and the bottom perforate like this.
7. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: glass or Pyrex wafer.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Oxide Etch (AOE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, is obtained the dark upright opening that degree of depth 1-50 μ m is arranged.
4. the opposite side of wafer (downside) applies photo-resist, and the figure that contains the diaphragm definition, by the exposure of UV light, is transferred on the photo-resist.
5. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Oxide Etch (AOE of Inductive Coupled Plasma (ICP), advanced silicon etching), wafer is carried out anisotropic etching, obtain vertical cavity at the wafer downside.The glass on etched timing definition wafer top or Pyrex residue diaphragm-operated thickness.
6. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
We do not enumerate the manufacture craft with chip glass.
(b) example on the plane surface of doing to revise on the surface with ICP and LPCVD, is manufactured with the method (Figure 11) of the long aperture that stretches out cocked bead
1. Kai Shi substrate: silicon single crystal wafer, crystallization direction＜100 〉.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, is obtained the dark upright opening that degree of depth 1-50 μ m is arranged.
4. with Low Pressure Chemical Vapour Deposition (LPCVD, low pressure chemical vapor deposition) or Plasma Enhanced Chemical Vapour Deposition (PECVD, the plasma-reinforced chemical vapour deposition), at silicon face coating silicon nitride.
5. the opposite side of wafer (downside) applies photo-resist, and the diaphragm-operated figure that contains perforate in the definition silicon nitride, by the exposure of UV light, is transferred on the photo-resist.
6. at the downside of wafer,, press the zone of perforate definition in the photo-resist, silicon nitride etch is fallen with Reaction Ion Etch (RIE, reactive ion etching).
7. in KOH or TMAH solution, the etching of wafer is anisotropic, produces the pyramid perforate at the wafer downside.Etched timing definition wafer upside excess silicon diaphragm-operated thickness.In addition, can mix with boron and define etched termination, can provide gauge control preferably.
8. at rear side RIE, remove the silicon nitride mask on the substrate rear side, and the rear end of opening aperture.
9. at front side RIE, remove the silicon nitride on the substrate front side, on opening, stay the silicon nitride edge of protrusion.
10. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: silicon single crystal wafer.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, is obtained the dark upright opening that degree of depth 1-50 μ m is arranged.
4. with Low Pressure Chemical Vapour Deposition (LPCVD, low pressure chemical vapor deposition) or Plasma Enhanced Chemical Vapour Deposition (PECVD, the plasma-reinforced chemical vapour deposition), at silicon face coating silicon nitride.
5. the opposite side of wafer (downside) applies photo-resist, and the diaphragm-operated figure that contains perforate in the definition silicon nitride, by the exposure of UV light, is transferred on the photo-resist.
6. at the downside of wafer,, press the zone of perforate definition in the photo-resist, silicon nitride etch is fallen with Reaction Ion Etch (RIE, reactive ion etching).
7. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), wafer is carried out anisotropic etching, produce the cylindricality perforate at the wafer downside.Etched timing definition wafer upside excess silicon diaphragm-operated thickness.
8. at rear side RIE, remove the silicon nitride mask on the substrate rear side, and the rear end of opening aperture.
9. at front side RIE, remove the silicon nitride on the substrate front side, on opening, stay the silicon nitride edge of protrusion.
10. or with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), perhaps use LPVCD, on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: the silicon (SOI) on the spacer of buried oxide layer is arranged, and zone of oxidation is at the following 1-50 μ of upper surface m place, carrier crystal orientation＜100 〉.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, obtain dark upright opening, the degree of depth is until the zone of oxidation of burying underground.
4. with Low Pressure Chemical Vapour Deposition (LPCVD, low pressure chemical vapor deposition) or Plasma Enhanced Chemical Vapour Deposition (PECVD, the plasma-reinforced chemical vapour deposition), at silicon face coating silicon nitride.
5. the opposite side of wafer (downside) applies photo-resist, and the diaphragm-operated figure that contains perforate in the definition silicon nitride, by the exposure of UV light, is transferred on the photo-resist.
6. at the downside of wafer,, press the zone of perforate definition in the photo-resist, silicon nitride etch is fallen with Reaction Ion Etch (RIE, reactive ion etching).
7. in KOH or TMAH solution, the etching of wafer is anisotropic, produces the pyramid perforate at the wafer downside.The zone of oxidation of burying underground will be as the etched termination of this process, and therefore, the thickness of silicon layer upside has defined residue diaphragm-operated thickness.
8. by RIE, moist hydracid (HF) etching or the HF vapor etch of fluoridizing, remove the exposed region of the zone of oxidation of burying underground.Can guarantee contacting between wafer middle and upper part and the bottom perforate like this.
9. at rear side RIE, remove the silicon nitride mask on the substrate rear side, and the rear end of opening aperture.
10. at front side RIE, remove the silicon nitride on the substrate front side, on opening, stay the silicon nitride edge of protrusion.
11. or, perhaps use LPVCD with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), on silicon, apply silicon oxide.
In addition, substrate can be made by following technology:
1. Kai Shi substrate: the silicon (SOI) on the spacer of buried oxide layer is arranged, and zone of oxidation is at the following 1-50 μ of upper surface m place.
2. silicon surface applied photo-resist, and handle contains the figure of aperture position and diameter, by the exposure of UV light, is transferred on the photo-resist.
3. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), the aperture graph transfer printing to silicon, obtain dark upright opening, the degree of depth is until the zone of oxidation of burying underground.
4. with Low Pressure Chemical Vapour Deposition (LPCVD, low pressure chemical vapor deposition) or Plasma Enhanced Chemical Vapour Deposition (PECVD, the plasma-reinforced chemical vapour deposition), at silicon face coating silicon nitride.
5. the opposite side of wafer (downside) applies photo-resist, and the diaphragm-operated figure that contains perforate in the definition silicon nitride, by the exposure of UV light, is transferred on the photo-resist.
6. at the downside of wafer,, press the zone of perforate definition in the photo-resist, silicon nitride etch is fallen with Reaction Ion Etch (RIE, reactive ion etching).
7. with Deep Reactive Ion Etch (DRIE, the deep reaction ion etching) or use the Advanced Silicon Etch (ASE of Inductive Coupled Plasma (ICP), advanced silicon etching), wafer is carried out anisotropic etching, produce vertical cavity at the wafer downside.The zone of oxidation of burying underground will be as the etched termination of this process, and therefore, the thickness of silicon layer upside has defined residue diaphragm-operated thickness.
8. by RIE, moist hydracid (HF) etching or the HF vapor etch of fluoridizing, remove the exposed region of the zone of oxidation of burying underground.Can guarantee contacting between wafer middle and upper part and the bottom perforate like this.
9. at rear side RIE, remove the silicon nitride mask on the substrate rear side, and the rear end of opening aperture.
10. at front side RIE, remove the silicon nitride on the substrate front side, on opening, stay the silicon nitride edge of protrusion.
11. or, perhaps use LPVCD with the thermooxidizing of plasma-reinforced chemical vapour deposition (PECVD), on silicon, apply silicon oxide.
Reference
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Neher，E.(2001).Molecular?biology?meets?microelectronics.Nature?Biotechnology?19：114
Penner，R.(1995).A?practical?guide?to?patch?clamping.In：Single-Channel?Recording.(Ed.E.Neher)Plenum?Press，New?Pork，London.
Rae，JL?and?Levis，RA(1992).Glass?technology?for?patch?clampelectrodes.Methods?Enzymol.207：66-92.
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Claims (29)
1. the substrate of planar basically that uses in the patch clamp, be used to analyze the cytolemma character that comprises glycocalix, wherein, this substrate includes the aperture at the edge of definition aperture, this edge is fit to form gigohm nurse sealing coat when contacting with cytolemma.
2. according to the planar substrates of claim 1, edge wherein protrudes from substrate plane, and the height of protrusion surpasses the thickness of glycocalix.
3. according to the planar substrates of claim 1 or 2, edge wherein protrudes from substrate plane, the height that protrudes is at least at the above 20nm in planar substrates surface, preferably 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm or 100nm at least at least at least at least at least at least at least at least.
4. according to each planar substrates of front claim, the width at edge wherein, scope 50 to 200nm.
5. according to each planar substrates of front claim, wherein the length of aperture (being the degree of depth) between 2 and 30 μ m, is preferably between 2 and 20 μ m, 2 and 10 μ m or the 5 and 10 μ m.
6. according to each planar substrates of front claim, the diameter of aperture wherein is in the scope of 0.5 to 2 μ m.
7. according to each planar substrates of front claim, edge wherein is substantially perpendicular to substrate plane and stretches out.
8. according to each substrate, edge wherein and substrate plane bevel of front claim 1 to 6.
9. according to each substrate of front claim 1 to 6, edge wherein is arranged essentially parallel to substrate plane.
10. according to the substrate of claim 1, edge wherein is by the mouth definition of aperture, this mouthful have 5 and 100nm between radius-of-curvature, have the angles of 45 to 90 degree.
11. according to each planar substrates of front claim, substrate wherein is by silicon, plastics, pure quartz or other glass, as quartz and PyrexTM, or mixed that one or more are selected from the quartz of following hotchpotch: Be, Mg, Ca, B, Al, Ga, Ge, N, P, As make.
12. according to the planar substrates of claim 11, substrate is wherein made by silicon.
13. according to each substrate of front claim, the substrate surface wherein and/or the wall of aperture have applied second kind of coating material.
14. according to the substrate of claim 13, coating material wherein is silicon, plastics, pure quartz, other glass, as quartz and PyrexTM, or mixed one or more be selected from the quartz of following hotchpotch: Be, Mg,
15. according to the substrate of claim 11, coating material wherein is a silicon oxide.
16. a manufacturing is according to the method for each substrate of claim 1 to 15, this method comprises the steps:
(i) provide the substrate template;
(ii) in template, form aperture; With
(iii) form the edge around aperture.
17. according to the method for claim 16, substrate is wherein made with silicon micro production technology.
18. according to the method for claim 16 or 17, step wherein (ii) comprises with inductive couple plasma (ICP) deep reactive ion etch process and forms aperture.
19., also comprise the step on coated substrates surface according to each method of claim 16 to 18.
20. according to the method for claim 19, step wherein (iii) is to carry out in coated substrates.
21. according to the method for claim 19, step wherein (iii) includes the direction ground and the intermediate steps of etch substrate front side selectively, removing the mask layer of substrate front side, and further the distance of the salient that has illustrated is continued to enter following substrate.
22. according to claim 19,20 or 21 method, coating wherein is with plasma-reinforced chemical vapour deposition (PECVD) and/or with low pressure chemical vapor deposition (LPCVD) deposit.
23. according to the method for claim 22, coating wherein is with plasma-reinforced chemical vapour deposition (PECVD) deposit.
24. according to the method for claim 18, step wherein (iii) comprises with plasma-reinforced chemical vapour deposition (PECVD), forms an edge from the coating of deposition surface.
25. a method that is used to analyze the electric physiological property that comprises the glycocalix cytolemma, this method comprises following step:
(i) provide substrate according to the marginate aperture of claim 1 to 15;
(ii) make cytolemma and substrate openings edge contact, so that between cytolemma and substrate, form gigohm nurse sealing coat; With
(iii) measure the electric physiological property of cytolemma.
26. a cover is used to implement instrument according to the method for claim 25, this cover instrument comprise according to claim 1 to 15 each substrate and a kind of or
27. one kind basically as the substrate of this paper front with reference to description of drawings.
28. one kind basically as the method for this paper front with reference to description of drawings.
29. a cover is basically as the instrument of this paper front with reference to description of drawings.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
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| US37279602P | 2002-04-17 | 2002-04-17 | |
| US60/372,796 | 2002-04-17 | ||
| GB0303922A GB2398635A (en) | 2003-02-21 | 2003-02-21 | A substrate providing a cell gigaseal for a patch clamp |
| GB0303922.9 | 2003-02-21 |
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| CN1646679A true CN1646679A (en) | 2005-07-27 |
| CN100506969C CN100506969C (en) | 2009-07-01 |
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| EP (1) | EP1495105B1 (en) |
| JP (1) | JP4351073B2 (en) |
| CN (1) | CN100506969C (en) |
| AT (1) | ATE354635T1 (en) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405410A (en) * | 2009-04-20 | 2012-04-04 | 牛津楠路珀尔科技有限公司 | Lipid bilayer sensor array |
| CN1987459B (en) * | 2006-12-14 | 2012-07-18 | 上海交通大学 | Method for producing biological sensor |
| CN104024839A (en) * | 2011-12-20 | 2014-09-03 | 独立行政法人科学技术振兴机构 | Planar Patch Clamp Device, Electrodes For Said Device And Cell Ion Channel Current Measurement Method |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2398635A (en) | 2003-02-21 | 2004-08-25 | Sophion Bioscience As | A substrate providing a cell gigaseal for a patch clamp |
| EP1801586B1 (en) * | 2002-08-21 | 2010-10-13 | Cellectricon Ab | System for obtaining and maintaining high-resistance seals in patch clamp recordings |
| JP4386835B2 (en) | 2002-08-21 | 2009-12-16 | セレクトリコン アーベー | System and method for acquiring and maintaining high resistance sticking in patch clamp recording |
| WO2007108773A1 (en) * | 2006-03-23 | 2007-09-27 | Agengy For Science, Technology And Research | Device for analyzing the status of a particle |
| JP4596009B2 (en) * | 2006-05-25 | 2010-12-08 | パナソニック株式会社 | Cell electrophysiological sensor chip, cell electrophysiological sensor using the chip, and method for manufacturing cell electrophysiological sensor chip |
| WO2010016193A1 (en) * | 2008-08-04 | 2010-02-11 | パナソニック株式会社 | Chip for cell electrophysiology sensor, cell electrophysiology sensor using same, and production method of chip for cell electrophysiology sensor |
| KR101119381B1 (en) * | 2009-06-18 | 2012-03-07 | 한국과학기술원 | Device and system for measuring properties of cells and method of measuring properties of cells using the same |
| JP5159808B2 (en) * | 2010-02-09 | 2013-03-13 | 日本電信電話株式会社 | Lipid bilayer substrate |
| CA2804564A1 (en) | 2010-07-09 | 2012-01-12 | Sophion Bioscience A/S | A chip assembly for use in a microfluidic analysis system |
| US9581562B2 (en) | 2011-03-01 | 2017-02-28 | Sophion Bioscience A/S | Handheld device for electrophysiological analysis |
| DE102013007295B4 (en) * | 2013-04-26 | 2015-06-25 | Universität Rostock | Electrophysiological measuring arrangement and electrophysiological measuring method |
| DE102014203280B4 (en) * | 2014-02-24 | 2015-12-17 | Cytocentrics Bioscience Gmbh | Device for determining measured quantities on membranes |
| JP7436991B2 (en) | 2021-02-24 | 2024-02-22 | 日本電信電話株式会社 | Lipid phase separation domain forming substrate and its manufacturing method, and substrate for lipid phase separation domain formation |
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| DE29721359U1 (en) * | 1997-12-03 | 1998-02-12 | Deutsche Itt Industries Gmbh, 79108 Freiburg | Device for measuring physiological parameters |
| AU746580B2 (en) * | 1997-12-17 | 2002-05-02 | Ecole Polytechnique Federale De Lausanne | Positioning and electrophysiological characterization of individual cells and reconstituted membrane systems on microstructured carriers |
| CA2374436A1 (en) * | 1999-05-21 | 2000-11-30 | James J. Hickman | Method of high throughput functional genomics comprising the analysis of the electrophysiology of neuronal cells |
| WO2001025769A2 (en) * | 1999-10-01 | 2001-04-12 | Sophion Bioscience A/S | A substrate and a method for determining and/or monitoring electrophysiological properties of ion channels |
| GB9930718D0 (en) * | 1999-12-24 | 2000-02-16 | Central Research Lab Ltd | Apparatus for and method of making electrical measurements on objects |
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2003
- 2003-04-17 AT AT03722763T patent/ATE354635T1/en not_active IP Right Cessation
- 2003-04-17 CA CA002480338A patent/CA2480338C/en not_active Expired - Fee Related
- 2003-04-17 AU AU2003229926A patent/AU2003229926A1/en not_active Abandoned
- 2003-04-17 DE DE60311973T patent/DE60311973T2/en not_active Expired - Lifetime
- 2003-04-17 US US10/511,320 patent/US20050212095A1/en not_active Abandoned
- 2003-04-17 WO PCT/GB2003/001705 patent/WO2003089564A1/en active IP Right Grant
- 2003-04-17 JP JP2003586275A patent/JP4351073B2/en not_active Expired - Lifetime
- 2003-04-17 DK DK03722763T patent/DK1495105T3/en active
- 2003-04-17 EP EP03722763A patent/EP1495105B1/en not_active Expired - Lifetime
- 2003-04-17 CN CNB038085569A patent/CN100506969C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1987459B (en) * | 2006-12-14 | 2012-07-18 | 上海交通大学 | Method for producing biological sensor |
| CN102405410A (en) * | 2009-04-20 | 2012-04-04 | 牛津楠路珀尔科技有限公司 | Lipid bilayer sensor array |
| CN102405410B (en) * | 2009-04-20 | 2014-06-25 | 牛津楠路珀尔科技有限公司 | Lipid bilayer sensor array |
| CN104024839A (en) * | 2011-12-20 | 2014-09-03 | 独立行政法人科学技术振兴机构 | Planar Patch Clamp Device, Electrodes For Said Device And Cell Ion Channel Current Measurement Method |
| CN104024839B (en) * | 2011-12-20 | 2016-11-16 | 国立研究开发法人科学技术振兴机构 | Planar diaphragm clamp device, this device electrode portion and cell ion channel current measuring method |
Also Published As
| Publication number | Publication date |
|---|---|
| DK1495105T3 (en) | 2007-06-04 |
| EP1495105B1 (en) | 2007-02-21 |
| AU2003229926A1 (en) | 2003-11-03 |
| DE60311973D1 (en) | 2007-04-05 |
| CA2480338A1 (en) | 2003-10-30 |
| DE60311973T2 (en) | 2007-10-31 |
| EP1495105A1 (en) | 2005-01-12 |
| CN100506969C (en) | 2009-07-01 |
| WO2003089564A1 (en) | 2003-10-30 |
| JP4351073B2 (en) | 2009-10-28 |
| CA2480338C (en) | 2009-07-07 |
| ATE354635T1 (en) | 2007-03-15 |
| US20050212095A1 (en) | 2005-09-29 |
| JP2005523011A (en) | 2005-08-04 |
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